Journal: Frontiers in Immunology

Article Title: Bone marrow mesenchymal stem cells alleviate smoke inhalation injury by regulating alveolar macrophage polarization via the CD200-CD200R pathway

doi: 10.3389/fimmu.2025.1645460

Figure Lengend Snippet: Expression levels of CD200 and CD200R on the surface of BMSCs and NR8383 cells and the effect of siRNA transfection of BMSCs on CD200 expression levels. (A) Immunofluorescence detection of positive CD200 expression on the surface of BMSCs (DAPI, blue; CD200, green); (B) Positive rate of CD200R expression on the surface of the NR8383 cell line detected by FCM, n= 3; (C) The transfection efficiency of siRNA at 24 h and 48 h was observed by immunofluorescence; (D, E) qPCR and WB analysis of CD200 mRNA and protein expression levels in BMSCs 48 h after transfection, n=3. ** P < 0.01, *** P < 0.001, ****P < 0.0001.

Article Snippet: Following blocking, the membranes were rinsed with TBST and then incubated overnight at 4°C with primary antibodies diluted in 5% skim milk (iNOS (ab178945, Abcam, USA), CD86 (30691-1-AP, Proteintech, China), CD200 (30570-1-AP, Proteintech, China), TGF-β (26155-1-AP, Proteintech, China), Arg-1 (16001-1-AP, Proteintech, China), CD206 (18704-1-AP, Proteintech, China), IL-β (26048-1-AP, Proteintech, China), p-P38 (4511S, Cell Signaling Technology, USA), P38 (8690S, Cell Signaling Technology, USA), p-JNK (4668S, Cell Signaling Technology, USA), JNK (ab179461, Abcam, USA), and GAPDH (60004-1-Ig, Proteintech, China)).

Techniques: Expressing, Transfection, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: Bone marrow mesenchymal stem cells alleviate smoke inhalation injury by regulating alveolar macrophage polarization via the CD200-CD200R pathway

doi: 10.3389/fimmu.2025.1645460

Figure Lengend Snippet: Effect of siCD200 on the M1-type polarization of alveolar macrophages. CD200 siRNA- or NC siRNA-transfected BMSCs were cocultured with M1-polarized NR8383 cells (NR8383 M1 ) at a ratio of 1:2, and the cells were collected after 24 h. (A) FCM assay for the percentage of iNOS + (M1-type) cells and Arg-1 + (M2-type) cells in NR8383 cells; (B–D) Quantitative analyses of the percentage of iNOS + cells, the percentage of Arg-1 + cells, and the iNOS + /Arg-1 + ratio in A; (E) WB detection of the expression of the M2-type markers CD206 and Arg-1 and secreted factor TGF-β and the M1-type marker iNOS and secreted factor IL-1β in NR8383 cells; (F-J) Quantitative analyses of the protein levels of TGF-β, Arg-1, CD206, IL-1β, and iNOS in (E) . Control levels were normalized to 1. The data are expressed as the means ± SDs. n=3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following blocking, the membranes were rinsed with TBST and then incubated overnight at 4°C with primary antibodies diluted in 5% skim milk (iNOS (ab178945, Abcam, USA), CD86 (30691-1-AP, Proteintech, China), CD200 (30570-1-AP, Proteintech, China), TGF-β (26155-1-AP, Proteintech, China), Arg-1 (16001-1-AP, Proteintech, China), CD206 (18704-1-AP, Proteintech, China), IL-β (26048-1-AP, Proteintech, China), p-P38 (4511S, Cell Signaling Technology, USA), P38 (8690S, Cell Signaling Technology, USA), p-JNK (4668S, Cell Signaling Technology, USA), JNK (ab179461, Abcam, USA), and GAPDH (60004-1-Ig, Proteintech, China)).

Techniques: Transfection, Expressing, Marker, Control

Journal: Frontiers in Immunology

Article Title: Bone marrow mesenchymal stem cells alleviate smoke inhalation injury by regulating alveolar macrophage polarization via the CD200-CD200R pathway

doi: 10.3389/fimmu.2025.1645460

Figure Lengend Snippet: BMSCs regulate alveolar macrophage polarization through the CD200-CD200R pathway via the inhibition of JNK-MAPK signaling. (A) NR8383 cells were treated with LPS/IFN-γ for 6 h, 12 h and 24 h, and the expression levels of pP38, P38, pJNK, JNK, and iNOS were detected by WB; (B) Quantitative analysis of the phosphorylation and protein levels in A; (C) NR8383 cells were pretreated with the JNK inhibitor SP600125 for 30 min before LPS/IFN-γ stimulation. After 24 h, the cells were collected, and the expression level of iNOS was detected by WB. (D) Quantitative analyses of protein levels in (C) . (E) NR8383 cells were pretreated with the JNK inhibitor SP600125 for 30 min before LPS/IFN-γ stimulation. After 24 h, the cells were collected, and the percentage of iNOS + (M1-type) cells was assayed by FCM. (F) Quantitative analysis of the percentages of iNOS + cells in (E) . (G) After being transfected with CD200 siRNA or NC siRNA, BMSCs were cocultured with M1-polarized NR8383 cells (NR8383 M1 ) at a 1:2 ratio for 24 h, and the effect of siCD200 on JNK phosphorylation was observed by WB. (H) Quantitative analyses of JNK phosphorylation levels in (E) . The control was normalized to 1. The data are expressed as the means ± SDs. n =3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following blocking, the membranes were rinsed with TBST and then incubated overnight at 4°C with primary antibodies diluted in 5% skim milk (iNOS (ab178945, Abcam, USA), CD86 (30691-1-AP, Proteintech, China), CD200 (30570-1-AP, Proteintech, China), TGF-β (26155-1-AP, Proteintech, China), Arg-1 (16001-1-AP, Proteintech, China), CD206 (18704-1-AP, Proteintech, China), IL-β (26048-1-AP, Proteintech, China), p-P38 (4511S, Cell Signaling Technology, USA), P38 (8690S, Cell Signaling Technology, USA), p-JNK (4668S, Cell Signaling Technology, USA), JNK (ab179461, Abcam, USA), and GAPDH (60004-1-Ig, Proteintech, China)).

Techniques: Inhibition, Expressing, Phospho-proteomics, Transfection, Control

Journal: Frontiers in Immunology

Article Title: Bone marrow mesenchymal stem cells alleviate smoke inhalation injury by regulating alveolar macrophage polarization via the CD200-CD200R pathway

doi: 10.3389/fimmu.2025.1645460

Figure Lengend Snippet: CD200 knockdown attenuates the therapeutic effect of BMSCs on lung tissue injury in rats with SII. (A) HE staining of lung tissues at 1 d and 3 d after BMSC transplantation (scale bar: 200 μm); (B, C) Lung injury scores at 1 d and 3 d after BMSC transplantation; (D, E) Wet/dry ratios of lung tissue at 1 d and 3 d after BMSC transplantation; (F, G) Total protein concentration in bronchoalveolar lavage fluid (BALF) at 1 d and 3 d after BMSC transplantation. n=5~6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following blocking, the membranes were rinsed with TBST and then incubated overnight at 4°C with primary antibodies diluted in 5% skim milk (iNOS (ab178945, Abcam, USA), CD86 (30691-1-AP, Proteintech, China), CD200 (30570-1-AP, Proteintech, China), TGF-β (26155-1-AP, Proteintech, China), Arg-1 (16001-1-AP, Proteintech, China), CD206 (18704-1-AP, Proteintech, China), IL-β (26048-1-AP, Proteintech, China), p-P38 (4511S, Cell Signaling Technology, USA), P38 (8690S, Cell Signaling Technology, USA), p-JNK (4668S, Cell Signaling Technology, USA), JNK (ab179461, Abcam, USA), and GAPDH (60004-1-Ig, Proteintech, China)).

Techniques: Knockdown, Staining, Transplantation Assay, Protein Concentration

Journal: Frontiers in Immunology

Article Title: Bone marrow mesenchymal stem cells alleviate smoke inhalation injury by regulating alveolar macrophage polarization via the CD200-CD200R pathway

doi: 10.3389/fimmu.2025.1645460

Figure Lengend Snippet: Effect of CD200 knockdown on the regulation of alveolar macrophage polarization by BMSCs in rats with SII. The cells isolated from BALF were blocked with an Fc receptor blocker for 15 minutes at 4°C. The samples were fixed and permeabilized using the Cyto-Fast™ Fix/Perm Buffer Set for 20 minutes. Then intracellular staining was performed for 30 minutes at room temperature in the dark. (A) FCM analysis of the percentages of alveolar macrophage (AM) iNOS + M1-type cells and Arg-1 + M2-type cells in the BALF 1 d after the transplantation of BMSCs. Alveolar macrophages were gated by CD68 marker as CD68 + . Then PE-anti-iNOS and FITC-anti-Arg-1 antibodies were used to analyze M1 and M2 cells, respectively. (E) FCM analysis of the percentages of iNOS + M1-type cells and CD206 + M2-type cells in alveolar macrophages (AMs) in the BALF 3 d after the transplantation of BMSCs. Alveolar macrophages were gated by CD68 marker as CD68 + . PE-anti-iNOS and FITC-anti-CD206 antibodies were used to analyze M1 and M2 cells, respectively. (B-D), (F-H) Quantitative analyses of (A, E) , respectively. The data are expressed as the means ± SDs. n=5~6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following blocking, the membranes were rinsed with TBST and then incubated overnight at 4°C with primary antibodies diluted in 5% skim milk (iNOS (ab178945, Abcam, USA), CD86 (30691-1-AP, Proteintech, China), CD200 (30570-1-AP, Proteintech, China), TGF-β (26155-1-AP, Proteintech, China), Arg-1 (16001-1-AP, Proteintech, China), CD206 (18704-1-AP, Proteintech, China), IL-β (26048-1-AP, Proteintech, China), p-P38 (4511S, Cell Signaling Technology, USA), P38 (8690S, Cell Signaling Technology, USA), p-JNK (4668S, Cell Signaling Technology, USA), JNK (ab179461, Abcam, USA), and GAPDH (60004-1-Ig, Proteintech, China)).

Techniques: Knockdown, Isolation, Staining, Transplantation Assay, Marker

Journal: Frontiers in Immunology

Article Title: Bone marrow mesenchymal stem cells alleviate smoke inhalation injury by regulating alveolar macrophage polarization via the CD200-CD200R pathway

doi: 10.3389/fimmu.2025.1645460

Figure Lengend Snippet: Effects of BMSCs on inflammatory factors in the BALF of rats with SII via CD200. (A-D) Expression levels of IL-1β, IL-6, TNF-α and TGF-β in the BALF 1 d after the transplantation of BMSCs. (E-H) IL-1β, IL-6, TNF-α, and TGF-β expression levels in BALF 3 days after transplantation of BMSCs. The data are expressed as the means ± SDs. n=5~6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Article Snippet: Following blocking, the membranes were rinsed with TBST and then incubated overnight at 4°C with primary antibodies diluted in 5% skim milk (iNOS (ab178945, Abcam, USA), CD86 (30691-1-AP, Proteintech, China), CD200 (30570-1-AP, Proteintech, China), TGF-β (26155-1-AP, Proteintech, China), Arg-1 (16001-1-AP, Proteintech, China), CD206 (18704-1-AP, Proteintech, China), IL-β (26048-1-AP, Proteintech, China), p-P38 (4511S, Cell Signaling Technology, USA), P38 (8690S, Cell Signaling Technology, USA), p-JNK (4668S, Cell Signaling Technology, USA), JNK (ab179461, Abcam, USA), and GAPDH (60004-1-Ig, Proteintech, China)).

Techniques: Expressing, Transplantation Assay